352 research outputs found
Translesion DNA synthesis-assisted non-homologous end-joining of complex double-strand breaks prevents loss of DNA sequences in mammalian cells
Double strand breaks (DSB) are severe DNA lesions, and if not properly repaired, may lead to cell death or cancer. While there is considerable data on the repair of simple DSB (sDSB) by non-homologous end-joining (NHEJ), little is known about the repair of complex DSBs (cDSB), namely breaks with a nearby modification, which precludes ligation without prior processing. To study the mechanism of cDSB repair we developed a plasmid-based shuttle assay for the repair of a defined site-specific cDSB in cultured mammalian cells. Using this assay we found that repair efficiency and accuracy of a cDSB with an abasic site in a 5′ overhang was reduced compared with a sDSB. Translesion DNA synthesis (TLS) across the abasic site located at the break prevented loss of DNA sequences, but was highly mutagenic also at the template base next to the abasic site. Similar to sDSB repair, cDSB repair was totally dependent on XrccIV, and altered in the absence of Ku80. In contrast, Artemis appears to be specifically involved in cDSB repair. These results may indicate that mammalian cells have a damage control strategy, whereby severe deletions are prevented at the expense of the less deleterious point mutations during NHEJ
The Metallo-β-Lactamase/β-CASP Domain of Artemis Constitutes the Catalytic Core for V(D)J Recombination
The V(D)J recombination/DNA repair factor Artemis belongs to the metallo-β-lactamase (β-Lact) superfamily of enzymes. Three regions can be defined within the Artemis protein sequence: (a) the β-Lact homology domain, to which is appended (b) the β-CASP region, specific of members of the β-Lact superfamily acting on nucleic acids, and (c) the COOH-terminal domain. Using in vitro mutagenesis, here we show that the association of the β-Lact and the β-CASP regions suffices for in vivo V(D)J recombination of chromosome-integrated substrates. Single amino acid mutants point to critical catalytic residues for V(D)J recombination activity. The results presented here define the β-Lact/β-CASP domain of Artemis as the minimal core catalytic domain needed for V(D)J recombination and suggest that Artemis uses one or two Zn(II) ions to exert its catalytic activity, like bacterial class B β-Lact enzymes hydrolyzing β-lactam compounds
Cernunnos/Xlf Deficiency Results in Suboptimal V(D)J Recombination and Impaired Lymphoid Development in Mice
Xlf/Cernunnos is unique among the core factors of the non-homologous end joining (NHEJ) DNA double strand breaks (DSBs) repair pathway, in the sense that it is not essential for V(D)J recombination in vivo and in vitro. Unlike other NHEJ deficient mice showing a SCID phenotype, Xlf−/− mice present a unique immune phenotype with a moderate B- and T-cell lymphopenia, a decreased cellularity in the thymus, and a characteristic TCRα repertoire bias associated with the P53-dependent apoptosis of CD4+CD8+ DP thymocytes. Here, we thoroughly analyzed Xlf−/− mice immune phenotype and showed that it is specifically related to the DP stage but independent of the MHC-driven antigen presentation and T-cell activation during positive selection. Instead, we show that V(D)J recombination is subefficient in Xlf−/− mice in vivo, exemplified by the presence of unrepaired DSBs in the thymus. This results in a moderate developmental delay of both B- and T-lymphocytes at key V(D)J recombination dependent stages. Furthermore, subefficient V(D)J recombination waves are accumulating during TCRα rearrangement, causing the typical TCRα repertoire bias with loss of distal Vα and Jα rearrangements
A new transcript in the TCRB locus unveils the human ortholog of the mouse pre-Dß1 promoter
Introduction: While most transcripts arising from the human T Cell Receptor locus reflect fully rearranged genes, several germline transcripts have been identified. We describe a new germline transcript arising from the human TCRB locus.
Methods: cDNA sequencing, promoter, and gene expression analyses were used to characterize the new transcript.
Results: The new germline transcript encoded by the human TCRB locus consists of a new exon of 103bp, which we named TRBX1 (X1), spliced with the first exon of gene segments C ss 1 or C ss 2. X1 is located upstream of gene segment D ss 1 and is therefore deleted from a V-DJ rearranged TCRB locus. The X1-C ss transcripts do not appear to code for a protein. We define their transcription start and minimal promoter. These transcripts are found in populations of mature T lymphocytes from blood or tissues and in T cell clones with a monoallelic TCRB rearrangement. In immature thymocytes, they are already detectable in CD1a(-)CD34(+)CD4(-)CD8(-) cells, therefore before completion of the TCRB rearrangements.
Conclusions: The X1 promoter appears to be the ortholog of the mouse pre-D ss 1 promoter (PD ss 1). Like PD ss 1, its activation is regulated by E ss in T cells and might facilitate the TCRB rearrangement process by contributing to the accessibility of the D ss 1 locus
Impaired Replication Stress Response in Cells from Immunodeficiency Patients Carrying Cernunnos/XLF Mutations
Non-Homologous End Joining (NHEJ) is one of the two major pathways of DNA Double Strand Breaks (DSBs) repair. Mutations in human NHEJ genes can lead to immunodeficiency due to its role in V(D)J recombination in the immune system. In addition, most patients carrying mutations in NHEJ genes display developmental anomalies which are likely the result of a general defect in repair of endogenously induced DSBs such as those arising during normal DNA replication. Cernunnos/XLF is a recently identified NHEJ gene which is mutated in immunodeficiency with microcephaly patients. Here we aimed to investigate whether Cernunnos/XLF mutations disrupt the ability of patient cells to respond to replication stress conditions. Our results demonstrate that Cernunnos/XLF mutated cells and cells downregulated for Cernunnos/XLF have increased sensitivity to conditions which perturb DNA replication. In addition, under replication stress, these cells exhibit impaired DSB repair and increased accumulation of cells in G2/M. Moreover Cernunnos/XLF mutated and down regulated cells display greater chromosomal instability, particularly at fragile sites, under replication stress conditions. These results provide evidence for the role of Cernunnos/XLF in repair of DSBs and maintenance of genomic stability under replication stress conditions. This is the first study of a NHEJ syndrome showing association with impaired cellular response to replication stress conditions. These findings may be related to the clinical features in these patients which are not due to the V(D)J recombination defect. Additionally, in light of the emerging important role of replication stress in the early stages of cancer development, our findings may provide a mechanism for the role of NHEJ in preventing tumorigenesis
16* Glutathione S-transferase activity-genotype association with time and severity of Pseudomonas aeruginosa lung infection in cystic fibrosis children
Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination
V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice
Immunologic aspects of patients with disseminated bacille Calmette-Guerin disease in north-west of Iran
<p>Abstract</p> <p>Background</p> <p>Adverse reactions induced by BCG vaccination are rare, disseminated mycobacterial BCG infection in particular, which is often fatal and results from impaired immunity. The aim of this study is to determine the nature of the immunodeficiences in patients with disseminated BCG infection in northwest region of Iran.</p> <p>Materials and methods</p> <p>Through 2 years all infants with BCG adenitis or other complications of this vaccine that had suspicious BCG infection were referred to children's hospital and health centers of Tabriz.</p> <p>Evaluation of immune system and in some cases genetic survey was performed in infants with evidence of histopathologic demonstration of acid-fast bacilli. Then frequency of infants who had disseminated BCG infection with immunodeficiency was defined.</p> <p>Results</p> <p>From 48 selected infants with complications of BCG vaccine in the range of 2 to 62 months, 28 infants (58.3%) were male and 20 infants (41.7%) were female. Disseminated BCG infection was diagnosed in 11 cases, almost all of whom had immunodeficiency as follows:</p> <p>Seven cases had severe combined immunodeficiency and one cases had chronic granulomatous disease. MSMD in two cases and IL12 R deficiency in another one was diagnosed.</p> <p>Overall, the mortality rate was 72.8% (8 cases) which 7 cases of them were SCID and another one CGD</p> <p>Consanguineous was found in more than half (7 cases) of patients and family history of disseminated BCG infection or immunodeficiency was found in nearly one third (3 cases) of patients.</p> <p>Discussion</p> <p>BCG vaccine is administered world wide to prevent tuberculosis and is considered to have excellent safety profile. However in some immunodeficient patients it can cause severe and fatal complications, like in our region, where all cases of disseminated BCG infection with severe immunodeficiency died.</p> <p>Conclusion</p> <p>BCG vaccination is necessary in some countries such as Iran, so it seems that development of a more safer vaccine and change of vaccine program in the families with history of inherited immunodeficiency can be identifies such high risk infants and prophylaxis of severe complications or dead in such patients.</p
A primary immunodeficiency characterized by defective immunoglobulin class switch recombination and impaired DNA repair
Immunoglobulin class switch recombination (CSR) deficiencies are rare primary immunodeficiencies, characterized by a lack of switched isotype (IgG, IgA, or IgE) production, variably associated with abnormal somatic hypermutation (SHM). Deficiencies in CD40 ligand, CD40, activation-induced cytidine deaminase, and uracil-N-glycosylase may account for this syndrome. We previously described another Ig CSR deficiency condition, characterized by a defect in CSR downstream of the generation of double-stranded DNA breaks in switch (S) μ regions. Further analysis performed with the cells of five affected patients showed that the Ig CSR deficiency was associated with an abnormal formation of the S junctions characterized by microhomology and with increased cell radiosensitivity. In addition, SHM was skewed toward transitions at G/C residues. Overall, these findings suggest that a unique Ig CSR deficiency phenotype could be related to an as-yet-uncharacterized defect in a DNA repair pathway involved in both CSR and SHM events
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